The present invention relates to biotechnology and more particularly to an apparatus for cultivating tissue cells or microorganisms in suspension.
An apparatus for cultivating tissue cells and microorganisms in suspension is known in the art (SU, A1, No. 1331888, 1987); which comprises a closed reservoir to the bottom of which branch pipes are connected for feeding an aerating gas. One group of the branch pipes is arranged tangentially, and the other parallel to the cylindrical wall of the reservoir. In the process of cultivating, the aerating gas at once involves the suspension in a vortex motion with simultaneous circulation in the form of ascending and descending streams along the axis of the apparatus.
However, such an apparatus cannot be used for cultivating cells of animal and human tissues: these cells can be easily traumatized, since in the process of cultivating in suspension a large number of gas bubbles is formed, and destruction of these bubbles will cause death of many cells. Aeration by gas blowing leads to the formation of foam which also results in loss of a part of the cells. Foam suppression will require introducing costly non-toxic chemical defoaming agents into the nutrient medium, the technological process of cultivation will be complicated, and the use of defoaming agents will deteriorate the properties of the culture medium.
An apparatus for cultivating tissue cells and microorganisms in suspension is known in the art (U.S. Pat. No. 4,259,449, 1981), which comprises a cylindrical reservoir with a cover and branch pipes for feeding an aerating gas and removing gaseous medium, and a device for stirring the cell suspension, said device being made as a grid disposed in the bottom part of the reservoir. Air is supplied through the grid to the reservoir for creating hydrostatic pressure which prevents precipitation of cells from the suspension.
However, the productivity of such an apparatus in the process of cultivating cells is low owing to worsening of the mass transfer characteristics which, in turn, are impaired, insofar as the intensity of feeding the aerating gas is reduced in order to obviate traumatizing the cells. Even in this case traumatizing the cells cannot be ruled out completely, and intensive foam formation takes place.
Also known in the art is an apparatus for cultivating tissue cells and microorganisms in suspension (WO 92/05245, A1, Feb. 4, 1992), comprising a cylindrical reservoir with a cover and branch pipes for gas feeding and removal, a device for aerating and stirring the suspension. The device for the aeration and stirring comprises a horizontal blade wheel secured on a vertical power shaft and arranged in the top part of the reservoir directly under the cover, and an annular plate placed thereunder, provided with a central opening for the removal of gas, attached over the periphery thereof to the wall of the reservoir, forming an annular space around the blade wheel for gas feeding and removal. Slot openings are provided in the annular partition for the passage of gas, said slots being distributed uniformly along the circumference at an angle to the horizontal plane. The branch pipe for feeding the gas is installed in the cover coaxially with the blade wheel, and the branch pipe for the removal of gas is coupled to said annular space and disposed on the edge of the cover.
The disadvantage of such an apparatus is that formation of vortex motion of liquid (potential vortex with axial reverse flow) therein can be achieved at high gas flow velocities (greater than 15-18 m/s) above the surface of this liquid, i.e., said vortex formation involves considerable power inputs. At the same time, there occurs entrainment of liquid drops from the surface of the suspension with subsequent ejection of these drops onto the wall of the reservoir. Cells in the suspension drops become traumatized by the impact against the wall of the apparatus, i.e., mass death of the cells takes place. With a decrease in the gas flow velocity (6-8 m/s), an unstable flow of the liquid above the surface of the cell suspension is observed, i.e., the axial-symmetric vortex motion of the liquid is periodically changed for auto-oscillations of the liquid, in which mode there originates a wave travelling along the wall of the reservoir. The surface of the liquid becomes curved and represents an asymmetric paraboloid of rotation. All the liquid in the apparatus oscillates as a single whole, the entire apparatus starts rocking, and this produces an unfavorable effect on the cell cultivation process. Besides, the design of the apparatus allows cultivating cells with the reservoir being filled to a height equal to or less than one diameter of this reservoir. If the reservoir is filled with the cell suspension to a height greater than one diameter thereof, a stagnant zone will be formed at the bottom of the reservoir. During cultivation cells will inevitably settle in this zone and perish because of deficiency in oxygen.
An apparatus for cultivating tissue cells or microorganisms in suspension is known, which comprises a cylindrical reservoir with a cover and branch pipes, accordingly, for feeding an aerating gas and removing gaseous medium, and a device for aerating and stirring the suspension, comprising a horizontal blade wheel secured on a vertical hollow shalt and arranged in the top part of the reservoir directly under the cover (WO 93/21301, A1, Oct. 28, 1993xe2x80x94the first embodiment of the apparatus). The device for aerating and stirring the medium is provided with an annular partition installed in the cylindrical reservoir coaxially with the blade wheel, a clearance being formed between the cylindrical wall of the reservoir and the annular partition; the device is also provided with a mechanism for stabilizing the position of the annular partition with respect to the surface of the cell suspension. In accordance with this embodiment of the invention, the mechanism for stabilizing the position of the annular partition with respect to the surface of the cell suspension is made in the form of racks attached to the cover of the reservoir and to the annular partition with help of latches with a possibility of varying the position of the annular partition in relation to the height of the reservoir. The annular partition should be immersed to a depth Hxe2x89xa70.02(D1-D2), where
D1 is the diameter of the annular partition;
D2 is the diameter of the axial opening in the annular partition.
A disadvantage of this embodiment of the apparatus is as follows. In many cases the process of cultivating cells and tissues is accompanied by changes in the level of the initial filling of the bioreactor (for example, owing to periodic sampling or the cultivation in the initial period being carried out with small volumes of the nutrient medium, whereas the final stage of cultivating is carried out with maximum filling of the reservoir of the reactor). As a result, the depth (H) of immersing the annular partition changes, i.e., the condition Hxe2x89xa7xe2x89xa70.02(D1-D2) is disturbed. A decrease of the value (H) results in xe2x80x9clockingxe2x80x9d of the liquid drain through the axial opening in the annular partition, and this leads to deterioration of the process of stirring and aerating the cells being cultivated. Should the annular partition hang stationary above the surface of the cell suspension, the mass-transfer parameters will worsen still further. If the depth of immersing (H) of the annular partition is appreciably greater than H=0.02(D1-D2), a travelling wave will be formed above the surface of the suspension, which gradually results in rocking the whole mass of the cell suspension, bringing it to the state of unstable stirring with lowered mass-transfer parameters. Therefore, under the conditions of periodic variations in the level of filling the reservoir of the apparatus in the process of cultivating, this device will be either almost inoperable, or introducing an additional mechanism will be required for automatic setting of the annular partition to the optimum depth on variations in the level of filling of the apparatus reservoir. This will make the apparatus much more complicated. Furthermore, the use of a stationary annular partition will involve additional power inputs and time expenditures for the bioreactor to reach the operating mode.
The known prior art most relevant to the proposed technical solution (prototype) is an apparatus for cultivating tissue cells or microorganisms in suspension, which comprises a cylindrical reservoir with a cover and branch pipes, accordingly, for feeding an aerating gas and removing gaseous medium, and a device for aerating and stirring the suspension, comprising a horizontal blade wheel secured on a vertical hollow shaft and arranged in the top part of the reservoir directly under the cover (WO 93/21301, A1, Oct. 28, 1993xe2x80x94the second embodiment of the apparatus). The device for aerating and stirring the medium is provided with an annular partition installed in the cylindrical reservoir coaxially with the blade wheel, a clearance being formed between the cylindrical wall of the reservoir and the annular partition; the device is also provided with a mechanism for stabilizing the position of the annular partition with respect to the surface of the cell suspension. The mechanism for stabilizing the position of the annular partition with respect to the surface of the cell suspension consists of floats with lead blades secured to the upper surface of the annular partition. The annular partition should be immersed to the depth Hxe2x89xa70.02(D1-D2), where
D1 is the diameter of the annular partition;
D2 is the diameter of the axial opening in the annular partition.
The disadvantage of this embodiment of the apparatus (the embodiment with the floating annular partition) is in that for reaching a high density of plant and animal cells in suspension or in cultivating highly aerobic cell cultures, the velocity of the air vortex above the surface of the liquid phase should be greater than 7-8 m/s in order to provide optimum conditions for aerating said biological objects. But with such velocities of the air vortex the intensity of the ascending fluid flow (axial reverse flow) is such that the annular partition floats up (because of the origin of pressure difference above and under the partition) to the surface of the cell suspension (the condition of optimum immersion of the annular partition Hxe2x89xa7xe2x89xa70.02(D1-D2) is disturbed, as a result of which the hydrodynamic mode of the flow (stirring) of the liquid (cell suspension) is disturbed, the conditions of aerating the cells being cultivated are worsened, this being followed by the precipitation of the biomass to the bottom of the reactor and death of the cells because of shortage in oxygen. On variations in the viscosity of the liquid phase in the process of cultivating biological objects, the annular partition, owing to its constant buoyancy, may uncontrollably change its position in relation to the surface of the liquid phase.
Besides, as the floating annular partition rotates in the cell suspension, stagnant shadow zones originate behind the floats, in which the cells being cultivated precipitate and accumulate. The lower layers of the cells perish in said zones because of shortage in oxygen, the quality of the finished product being thereby impaired and the technological characteristics of the apparatus lowered.
The present invention is directed to the provision of an apparatus for cultivating tissue cells or microorganisms in suspension which would ensure formation of an axial-symmetric vortex motion of liquid with axial reverse flow in the cell suspension without stagnant zones either at low velocities of gas movement (3-6 m/s) or at higher velocities thereof (7-10 m/s and more) above the surface of this liquid due to maintaining the annular partition at the optimal depth irrespective of changes in the intensity of the gas vortex above the surface of the cell suspension and changes in the viscosity of the liquid phase, which, in turn, will allow cultivating tissue cells or microorganisms having different oxygen requirements.
The set object is accomplished due to the fact that in an apparatus for cultivating tissue cells or microorganisms in suspension, which comprises a cylindrical reservoir with a cover and branch pipes, accordingly, for feeding an aerating gas and removing gaseous medium, and a device for aerating and stirring the suspension, comprising a horizontal blade wheel secured on a vertical hollow shaft and arranged in the top part of the reservoir directly under the cover, an annular partition installed in the cylindrical reservoir coaxially with the blade wheel with a clearance being formed between the cylindrical wall of the reservoir and the annular partition, and a mechanism for stabilizing the position of the annular partition with respect to the surface of the liquid phase (of the cell suspension), made in the form of guide elements and floats, according to the invention, the guide elements of the mechanism for stabilizing the position of the annular partition with respect to the surface of the liquid are made as detachable blades with a flat upper surface and a convex lower surface, said blades being oriented radially with respect to the annular partition and the surfaces of said blades defining an aerodynamic profile thereof of the xe2x80x9cforward-sweep wingxe2x80x9d type; the blades are attached with the help of struts to the upper surface of the annular partition or the blades are attached with the help of struts to the upper and lower surfaces of this partition, the blades being provided with units for varying the angle of attack with respect to the incoming flow of gas or liquid and for securing the blades to the struts on the annular partition and under it, respectively.
The aero- or hydrodynamic force originating as the blades of such design (with the profile of the xe2x80x9cforward-sweep wing type) are streamlined by gas or liquid, is directed against the hydrodynamic force causing floating-up of the annular partition, and this allows said annular partition to be kept at an optimum depth when the velocity of the gas vortex above the surface of the liquid phase is greater than 6-7 m/s.
The units for varying the angle of attack with respect to the incoming flow of gas or liquid and for securing the blades to the struts on the annular partition and under it are made as clamping devices. Said units can be made as various plug-type connections, for instance, of the screw-nut or collet type.
When the blades on the annular partition are disposed above the surface of the cell suspension, the angle of attack of the blades with respect to the incoming flow of gas is from xe2x88x9215xc2x0 to xe2x88x9290xc2x0, and when the blades are disposed in the liquid under the annular partition, the angle of attack of the blades with respect to the incoming flow of the liquid is from 0xc2x0 to xe2x88x9235xc2x0.
In said angular ranges of inclination of the blades to the incoming flow of gas or liquid stable retention of the annular partition at an optimal depth with low power consumption is ensured.
The floats of the mechanism for stabilizing the position of the annular partition with respect to the surface of the liquid phase are made in the body of this partition. They are shaped as truncated unequal-sided pyramids oriented with the truncated vertexes toward the annular partition and fixed on the struts with a clearance in relation to the blades and the annular partition.
Such configuration of the floats ensures an increase in the reliability of maintaining the annular partition at an optimal depth irrespective of variations in the viscosity of the liquid phase, while the clearance between the floats and the annular partition eliminates stagnation shadow zones behind the floats, whereby settling, accumulation and death of the cultivated cells in these zones are prevented.
Arranging the floats in the body of the annular partition also eliminates stagnant shadow zones on this partition, whereby settling, accumulation and death of the cultivated cells in these zones are obviated.
The annular partition is immersed into the cell suspension to depth (H) equal to H=0.02-0.09(D1-D2), where
D1 is the diameter of the annular partition;
D2 is the diameter of the axial opening in the annular partition.
A decrease in the value H less than 0.02(D1-D2) results in xe2x80x9clockingxe2x80x9d the drain of liquid through the axial opening in the annular partition, this leading to deterioration of the process of stirring and aerating the cultivated cells, with subsequent settling of the biomass to the bottom of the reactor and death of the cells because of shortage in oxygen. If the depth of immersion (H) of the annular partition is appreciably greater than H=0.09(D1-D2), a travelling wave is formed above the surface of suspension, which gradually brings the whole mass or the cell suspension into rocking and then into the mode of unstable stirring with lowered mass-transfer parameters (auto-oscillation mode). This negatively influences the viability and productivity of the biomass.